Streptococcus pneumoniae (pneumococcus) is one of the most potent human pathogens, affecting over 10 million people worldwide, of all age groups, in particular young children, the elderly and the immunocompromised. It is a leading causative agent of serious, often fatal diseases, such as pneumonia, bacteraemia and meningitis. It is also responsible of other less serious, but nevertheless debilitating diseases such as otitis media and keratitis.
Even after decades of using antibiotics and steroids as adjunctive to antibiotics the mortality and morbidity from pneumococcal diseases remains very high in the developed world and alarmingly high in the developing world. Nearly 20% of hospitalised patients still die despite antibiotic killing of the pneumococcus, while many survivors of pneumococcal meningitis suffer severe neurological handicaps, including cognitive impairment, vision and hearing loss, hence imposing huge distress on patients and their families and a very significant cost to healthcare systems. Today, infection with pneumococcus remains a major global public health problem that is widely recognised by leaders in the field and by health organisations, including the WHO.
One of the leading factors for this consistently high mortality and morbidity that is not addressed by the current standard therapy, is the toxaemia resulting from the release of toxic pneumococcal products, the most important of which is the pneumococcal toxin pneumolysin. This toxin is a major player in pneumococcal virulence and is the primary direct and indirect cause of toxaemia.
Pneumolysin belongs to the family of cholesterol dependent cytolysins (CDCs), which bind to cholesterol containing membranes and generate large pores that have lethal and sub-lethal effects on the affected cells. In the bacterium, the toxin pneumolysin is cytoplasmic and is mainly released from the pneumococcus after its lysis. Consequently, under the effect of lytic antibiotics, a large bolus of toxin is released, compounding the toxaemia. Thus, even if treatment with antibiotics is successful in clearing the bacteria from the patients, the subsequent release of the toxin is detrimental and can be fatal or cause long-term handicaps.
This toxaemia constitutes a substantial unmet medical need that is internationally recognised. Currently, corticosteroids, principally dexamethasone, are used as an adjunctive to antibiotic therapy for pneumococcal meningitis. However, even when dexamethasone is used, significant mortality and morbidity are seen and the widespread use of dexamathasone is still debated due to its non-specific effect, limited clinical impact and in some cases its detrimental effect in increasing neuronal apoptosis in meningitis [Lancet (2002) 360 211-218]. Therefore, the present state of the art is not adequate for the efficient treatment of invasive pneumococcal diseases.
There is considerable evidence substantiating the validity of pneumolysin as a therapeutic target. In the laboratories of the inventors it has been demonstrated that, using a mouse pneumonia model, a mutated strain of S. pneumoniae (PLN-A) that does not produce pneumolysin is no longer lethal, causes substantially less bacteraemia and exhibits a significant reduction in the severity of pulmonary inflammation. Other evidence obtained in a rat meningitis model, has shown that infection with the pneumolysin-negative mutant was markedly less severe than with wild-type pneumococci, with no observed damage to the ciliated epithelium of the brain and no apoptosis of the cells surrounding the epithelium [J. Infect, (2007) 55 394-399]. In pneumococcal meningitis in guinea pigs, wild-type pneumococci induced severe cochlear damage and hearing loss, while infection with PLN-A left the organ of Corti intact [Infect. Immun. (1997) 65 4411-4418]. An ex vivo model using cultured ciliated brain epithelial cells, enabled recreation of the in vivo situation, where cells lining the brain ventricles are exposed to S. pneumoniae. Both intact and antibiotic-killed wild-type pneumococci induced damage to the epithelial cells in culture and significantly impaired ciliary beating; effects not seen with PLN-A [Infect. Immun. (2000) 68 1557-1562]. This damaging effect of antibiotic-lysed pneumococci on the cultured ependymal cells is clearly caused by the toxin pneumolysin released from the antibiotic-lysed bacteria, as this damage was abolished in the presence of anti-pneumolysin antibodies [Infect. Immun. (2004) 72 6694-6698]. This finding supports the strategy that antibiotic-induced toxaemia is prevented by combination with anti-pneumolysin agents.
Evidence for the significant involvement of pneumolysin in pneumococcal infections and the substantial improvement of the disease prognosis in the absence of pneumolsyin, has led to the conclusion that pneumolysin constitutes a potential therapeutic target to develop new treatments for pneumococcal diseases. Previous research has shown the ability of cholesterol to inhibit pneumolysin [Biochem. J. (1974) 140 95-98], however, this inhibition is merely due to the fact that cholesterol is a natural cellular receptor of pneumolysin that is required for the pore formation in the target cell membrane. The topical application of cholesterol on the cornea of rabbits demonstrated a positive therapeutic effect in pneumococcal keratitis [Invest. Ophtalmol. Vis. Sci. (2007) 48 2661-2666]. This indicates the involvement of pneumolysin in pneumococcal keratitis and the therapeutic benefit obtained following its inhibition. However, cholesterol is not considered as a therapeutic agent for the treatment of pneumococcal diseases and has not been clinically used in patients. Another pneumolysin inhibitor, Allicin, a component in garlic extract, has been previously found to inhibit the haemolytic activity of pneumolysin in vitro [Toxicon (2011) 57 540-545]. This compound is a cysteine inhibitor that irreversibly binds to the reactive thiol group of the toxin. Compounds exhibiting such a property are unfavourable as drug candidates because of their potential unspecific binding to other cysteine-containing proteins in the body.
There remains a need to provide inhibitors of cytolysins, such as pneumolysin, which are suitable for use in the treatment of bacterial infections.
The present invention provides compounds that specifically inhibit the direct toxic effect of pneumolysin and other cholesterol dependent cytolysins that are pivotal in the virulence of their respective hosts. The compounds of the invention have no structural similarity to Allicin and do not bind covalently to the reactive thiol groups of the toxins.
Certain N-phenyl substituted pyrroles are known, however their use as pharmaceuticals in particular for the treatment of bacterial infections had not been suggested. The compounds diethyl 3,4-dihydroxy-1-(4-methoxyphenyl)-1H-pyrrole-2,5-dicarboxylate (CAS 654052-34-3) and diethyl 3,4-dihydroxy-1-phenyl-1H-pyrrole-2,5-dicarboxylate (CAS 55932-13-3) are commercially available. The compounds dimethyl 3,4-dihydroxy-1-(4-bromophenyl)-1H-pyrrole-2,5-dicarboxylate (CAS 1087699-40-8), dimethyl 3,4-dihydroxy-1-(4-chlorophenyl)-1H-pyrrole-2,5-dicarboxylate (CAS 1082655-47-7), di-tert-butyl 3,4-dihydroxy-1-(4-nitrophenyl)-1H-pyrrole-2,5-dicarboxylate (CAS 110332-46-2) and dimethyl 3,4-dihydroxy-1-(4-methoxyphenyl)-1H-pyrrole-2,5-dicarboxylate (CAS 101090-98-6) are described in Justus Liebigs Annalen der Chemie (1961), 639, 102-24. The compounds dimethyl 3,4-dihydroxy-1-phenyl-1H-pyrrole-2,5-dicarboxylate (CAS 7803-73-8) and dimethyl 3,4-bis(acetyloxy)-1-phenyl-1H-pyrrole-2,5-dicarboxylate (CAS 7342-22-5) are described in Hoppe-Seyler's Zeitschrift fuer Physiologische Chemie (1956), 306, 49-55. The compound diethyl 3,4-bis(acetyloxy)-1-phenyl-1H-pyrrole-2,5-dicarboxylate (CAS 55932-14-4) is disclosed in Chemische Berichte (1975), 108(2), 569-75.
The compounds of the present invention also prevent stimulation of host-derived toxic effects induced by pneumolysin and other cholesterol dependent cytolysins. Thus these compounds may be used as single agents or as adjunct to antibiotics, to prevent or attenuate pneumolysin-induced toxicity and its anti-host effects seen during infections caused e.g. by S. pneumoniae. 